The three dimensional structure of RNA is critical to its cellular function. For example, catalytic RNAs, known as ribozymes, can fold into complex globular structures and promote chemical reactions at specific sites with substantial rate enhancements independent of protein facilitation. Several other RNAs important to cellular viability are also precisely structured including rRNA, tRNA, and regulatory regions within mRNA. Thus, like proteins, there are important questions about RNA structure and the mechanism by which they promote catalysis. Noncanonical base pairs such as G.A, G.G, U.C, and G.U are critical to the higher order structure and function of RNA. The most frequently encountered noncanonical base pair is the G.U wobble pair, wherein G makes two hydrogen bonds with U in a geometry shifted from the standard Watson-Crick configuration. Phylogenetically conserved G.U wobble pairs are located in most classes of structured RNAs including several in rRNA, tRNA/A1a, and the catalytic domains of many ribozymes. Noncanonical base pairs appear to be a common strategy to promote defined RNA tertiary structures by providing a unique array of functional groups for hydrogen bonding in both the major and minor grooves of the RNA helix. The aim of this proposal is to define the basis for noncanonical base pair conservation and its role in the tertiary folding of RNA. Functional groups in noncanonical pairs will be identified that play an important role in RNA structure and function. Such a characterization cannot be carried out simply by mutagenesis because stable pairing combinations between the natural bases change the base pair to a Watson-Crick conformation and thereby alter the structural context of the functional groups. Synthetic organic chemistry will be used to prepare nucleotide analogs that systematically alter the functional groups of the bases, but retain the pairing conformation.